Document [original]

Investigating the role of GLUL as

a survival factor in cellular

adaptation to glutamine

depletion via targeted stable

isotope resolved metabolomics

Șafak Bayram

1†

, Yasmin Sophiya Razzaque

1†

Sabrina Geisberger

1†

, Matthias Pietzke

, Susanne Fürst

Carolina Vechiatto

, Martin Forbes

, Guido Mastrobuoni

and

Stefan Kempa

Proteomics and Metabolomics Platform, Max-Delbrück-Center for Molecular Medicine (MDC), Berlin

Institute for Medical Systems Biology (BIMSB), Berlin, Germany,

Mass Spectrometry Facility, Max

Planck Institute for Molecular Genetics, Berlin, Germany,

Theoretical Chemistry Quantum Chemistry,

Institute for Chemistry, Technische Universität Berlin, Berlin, Germany

Cellular glutamine synthesis is thought to be an important resistance factor in

protecting cells from nutrient deprivation and may also contribute to drug

resistance. The application of ‟targeted stable isotope resolved metabolomics”

allowed to directly measure the activity of glutamine synthetase in the cell. With

the help of this method, the fate of glutamine derived nitrogen within the

biochemical network of the cells was traced. The application of stable isotope

labelled substrates and analyses of isotope enrichment in metabolic

intermediates allows the determination of metabolic activity and ﬂux in

biological systems. In our study we used stable isotope labelled substrates of

glutamine synthetase to demonstrate its role in the starvation response of

cancer cells. We applied

C labelled glutamate and

N labelled ammonium and

determined the enrichment of both isotopes in glutamine and nucleotide

species. Our results show that the metabolic compensatory pathways to

overcome glutamine depletion depend on the ability to synthesise glutamine

via glutamine synthetase. We demonstrate that the application of dual-isotope

tracing can be used to address speciﬁc reactions within the biochemical

network directly. Our study highlights the potential of concurrent isotope

tracing methods in medical research.

KEYWORDS

targeted stable isotope resolved metabolomics, GLUL, nucleotide biosynthesis,

glutamine addiction, cancer metabolism, glutamine synthetase

OPEN ACCESS

EDITED BY

Wolfram Weckwerth,

University of Vienna, Austria

REVIEWED BY

Mariafrancesca Scalise,

University of Calabria, Italy

Denise Toscani,

University of Parma, Italy

*CORRESPONDENCE

Stefan Kempa,

stefan.kempa@mdc-berlin.de

†

These authors have contributed equally

to this work

SPECIALTY SECTION

This article was submitted to

Metabolomics,

a section of the journal

Frontiers in Molecular Biosciences

RECEIVED 21 January 2022

ACCEPTED 15 July 2022

PUBLISHED 12 August 2022

CITATION

Bayram Ș, Razzaque YS, Geisberger S,

Pietzke M, Fürst S, Vechiatto C,

Forbes M, Mastrobuoni G and Kempa S

(2022), Investigating the role of GLUL as

a survival factor in cellular adaptation to

glutamine depletion via targeted stable

isotope resolved metabolomics.

Front. Mol. Biosci. 9:859787.

doi: 10.3389/fmolb.2022.859787

Pietzke, Fürst, Vechiatto, Forbes,

Mastrobuoni and Kempa. This is an

open-access article distributed under

the terms of the Creative Commons

Attribution License (CC BY). The use,

distribution or reproduction in other

forums is permitted, provided the

original author(s) and the copyright

owner(s) are credited and that the

original publication in this journal is

cited, in accordance with accepted

academic practice. No use, distribution

or reproduction is permitted which does

not comply with these terms.

Frontiers in Molecular Biosciences frontiersin.org01

TYPE Original Research

PUBLISHED 12 August 2022

DOI 10.3389/fmolb.2022.859787

1 Introduction

Reprogramming of cellular metabolism was the earliest

molecular phenotypes described in cancer cells; Otto Warburg

described the preference of cancer cells to ferment pyruvate into

lactic acid even in the presence of oxygen and Hanahan and

Weinberg ﬁnally included metabolic reprogramming into their

list of hallmarks of cancer (Warburg, 1956;Vander Heiden et al.,

2009;Hanahan and Weinberg, 2011).Nowadays more and more

detailed studies show a complex deregulation of cancer cell

metabolism that is connected to growth and proliferation,

immune cell evasion and also drug resistance mechanisms.

Despite a profound activation of glucose metabolism, cancer

cells metabolise amino acids, such as glutamine (Lieu et al., 2020).

Glutamine is a non-essential amino acid, and the most abundant

in human blood plasma. Besides providing a source of energy,

glutamine is required for several processes, including

macromolecule biosynthesis, amino acid uptake, inhibition of

autophagy and it triggers target of rapamycin (mTOR) kinase

activation (Nicklin et al., 2009). Glutamine derived carbon fuels

the tricarboxylic acid (TCA) cycle. The amino group of

glutamine contributes to the synthesis of non-essential amino

acids via the transaminase network and the amido-group serves

as an obligate nitrogen donor for de novo nucleotide synthesis

and hexosamine synthesis, speciﬁcally reactions that use the

amido-nitrogen make glutamine a conditional essential

metabolite (Altman et al., 2016;Bott et al., 2019).

Four different glutamine transport systems are characterized

so far. These are known as SNAT3 (System N, SLC38A3) which is

important in glutamine uptake in periportal cells in liver and in

renal proximal tuble cells. SNAT1 (SLC38A1) is important in

glutamine uptake by neuronal cells and ASCT2 (SLC1A5) is

essential for glutamine uptake by rapidly growing epithelial cells

and tumour cells in culture; the brush border membrane

transporter B0 AT1 (SLC6A19) facilitates the uptake of

glutamine across the kidney and intestinal brush border

(McGivan and Bungard, 2007).

In the absence of sufﬁcient extracellular glutamine,

intracellular de novo synthesis can provide this essential

metabolite. Glutamine synthetase (GS), also referred to as

glutamate-ammonia ligase (GLUL) ligates glutamate with

ammonia in an ATP-dependent condensation reaction

(Nicklin et al., 2009). Several studies have revealed that the

depletion of glutamine causes cell death (Eagle, 1955;Yuneva

et al., 2007). This phenomenon, termed glutamine addiction, has

been observed in a variety of cancer types in in vitro and in vivo

studies (Wise and Thompson, 2011). Additionally, the

reprogrammed metabolism of glutamine was shown to be

crucial in tumorigenesis and tumour development (Yoo et al.,

2020). Nevertheless, the molecular mechanisms underlying

glutamine addiction are still not fully resolved.

Recent studies have demonstrated that glutamine deprived

cells can be rescued by asparagine supplementation, for unclear

reasons (Zhang et al., 2014). In the absence of glutamine, cells

were rescued to a greater extent by asparagine supplementation

relative to α-ketoglutarate, aspartate or glutamate (Zhu et al.,

2017). In summary, asparagine has been demonstrated to

regulate cell growth and rescue glutamine deﬁciency via

several potential mechanisms (Zhang et al., 2014;Krall and

Christofk, 2015;Zhang et al., 2017;Zhu et al., 2017;Pavlova

et al., 2018). Understanding these mechanisms is fundamental to

the development and efﬁcacy of metabolic therapies targeting

asparagine and glutamine metabolism. Interestingly rat stem cells

transformed by the oncogenic Kaposi’s sarcoma-associated

herpesvirus (KSHV) demonstrated the capacity to utilise the

amido group from both glutamine and asparagine for purine and

pyrimidine biosynthesis (Zhu et al., 2017). In our study we have

shown that the ability of colon cancer cells to compensate

glutamine withdrawal by asparagine supplementation did

solely depend on intracellular de novo glutamine synthesis by

glutamine synthetase (GLUL).

Dejure and Royla examined the growth behaviour of a panel

of cell lines under glutamine supplemented and glutamine

depleted conditions (Dejure et al., 2017). All tested colon

cancer cells (HCT116, GEO, HT29, SW480, RKO) stopped

proliferation in glutamine depleted conditions, while

HEK293 cells were able to proliferate. Our investigations

revealed that the ability of HEK293 cells to proliferate in

glutamine deprived conditions was abolished when dialyzed

serum was used in the growth medium. Therefore, the

previously observed “glutamine independency”of

HEK293 cells may be attributed to remaining small molecules

enabling glutamine synthesis. To identify which amino acids

enable cell growth in glutamine depleted conditions, cell growth

assays were performed with supplementation of either glutamine,

asparagine, glutamate, aspartate and alanine with or without

ammonium. GLUL’s substrates, glutamate and ammonium, were

associated with the greatest proliferation rate in the absence of

glutamine in HEK293 and HCT116 cells. RKO cells were unable

to proliferate in the absence of glutamine. The application of a

competitive inhibitor of GLUL, methionine sulfoximine (MSO),

prevented proliferation in the absence of glutamine also in

HEK293 and HCT116 cells. Taken together, these ﬁndings

pointed towards a key role for GLUL in adaptation to

glutamine depletion.

To demonstrate GLUL activity and to determine the

metabolic fate of GLUL’s substrates, a dual-tracer and targeted

Stable Isotope Resolved Metabolomics (SIRM) method was

established. We developed a “targeted SIRM”dual isotope

tracing technique in which substrates speciﬁc to a biological

reaction are differentially labelled and monitored via high

resolution mass spectrometry. In this case, the simultaneous

application of

C-glutamate and

N-ammonium allowed us

to detect the relative contribution of extracellular glutamate

and ammonium to intracellular glutamine synthesis, as well as

monitor the downstream contribution of glutamine’s carbon and

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nitrogen to de-novo nucleotide biosynthesis. We also performed a

time resolved dual-isotope tracing analysis and found the kinetics

of glutamine synthesis in HEK293 and HCT116 cells are distinct,

RKO cells did not show de novo glutamine synthesis, although

the protein could be detected in proteomics analyses and western

blot experiments.

Furthermore, we present growth conditions that preserve the

viability of glutamine-dependent cancer cells under glutamine

depletion. Our data show that all different amino acid

supplementations that enable cell survival and proliferation

with or without ammonium depend ﬁnally on the intracellular

activity of GLUL. With the new established method of dual

tracing and targeted pulsed stable isotope resolved metabolomics

we could analyze the dynamics of intracellular glutamine

synthesis.

2 Materials and methods

2.1 Cell culture

The standard cell culture medium (glutamine-supplemented

medium) comprised Dulbecco’s Modiﬁed Eagle Medium

(DMEM, Thermo Fisher) without glucose (Glc), glutamine

(Gln), phenol red or sodium pyruvate, supplemented with

10% dialyzed fetal bovine serum (dFBS), 2.5 g/L Glc, and

2 mM Gln. HEK293, HCT116 and RKO cells were grown in

10 cm plates at 37°C, 5% CO

, 21% O

and 85% relative

humidity, and were passaged every 3,4 days to avoid contact

inhibition and supply new media. When a conﬂuency of at least

70% was reached, cells were washed once with 1x PBS and

detached from the plate via trypsinization with TrypLE

(GIBCO). Pre-warmed medium was added to cease

trypsinization. The volume of medium added was calculated

according to the desired splitting ratio and the cells were

resuspended before the appropriate fraction of the cell

suspension was transferred to a new plate. For cell growth

assays and subsequent experiments, cells were harvested at a

conﬂuency of 80%–90% before being transferred to new plates at

a seeding density of 2×10

cells which prevents contact

inhibition.

2.2 Cell growth analysis

For the cell growth assays, pre-cultivated cells were seeded on

10 cm plates. The following day, the viable cell count was

measured for the 0 hour time point and the cell culture

medium was changed to that containing the appropriate

condition (Gln: 2 mM; Alanine (Ala), Asparagine (Asn),

Aspartate (Asp), Glutamate (Glu): all 1 mM, NH

: 0.8 mM).

Cells were passaged once they reached a conﬂuency of at least

60%, upon which the cell count was determined. Media was

replaced every 3,4 days to avoid limiting nutrients. Viability and

cell number were monitored using the TC20 automated cell

counter (Biorad).

2.3 Methionine sulfoximine inhibitor

proliferation assay

Pre-cultivated cells were seeded on 6-well plates at a seeding

density of 3×10

cells and 12×10

cells for HCT116 and

HEK293 cells, respectively. The following day, the viable cell

count was measured for the 0 hour time point and the cell culture

medium was changed to that containing the appropriate culture

condition (see Section 2.1) treated with either 500 µM

Methionine Sulfoximine (MSO, Sigma Aldrich) or, as a

negative control, sterile water (H

O). The viable cell count

was determined at 24, 48, 72, and 96 h post-treatment. Media

was replaced daily to replenish substrates and remove secreted

reaction products.

2.4 Western blotting

Cells grown in standard media conditions (not starved for

glutamine) were washed with PBS and harvested in 1 ml ice-cold

RIPA buffer. Cell lysates of HCT116, RKO and HEK293 were

denatured in loading buffer for 5 min at 95°C. 40 µg of proteins

were loaded and separated on a 10% SDS gel and run for 1 h at

70 V and 1 h at 120 V. The gel was transferred to a nitrocellulose

membrane (0.2 µm, Biorad) at 25 V, 1 A for 30 min (Biorad

TransBlot Turbo V1.02). The membrane was blocked for 1.5 h in

5% milk in TBS-T at room temperature and cut below the 70 kDa

band. The membranes were incubated with primary antibodies

against Vinculin (1:2,000 dilution, Sigma, V9131) and against

GLUL (1:1,000 dilution, Thermo Fisher, PA1-46165) in 5% milk

in TBS-T over-night at 4°C. After washing the membranes in

TBST, the membranes were incubated in the HRP-conjugated

secondary antibodies (NEB, 7074S; NEB, 7076S) for 1 h at room

temperature. After washing the membranes in TBST and TBS,

the membrane was developed using an ECL Western Blotting

detection reagent (Amersham, RPN2109) according to the

manufacturer’s protocol. The Vilber FX gel system was used

to record the luminescence (Vilber Lourmat, France).

2.5 Targeted stable isotope resolved

metabolomics and pSIRM

Cells were pre-cultivated in Glu + NH

-supplemented

medium for at least 3 days prior to stable SIRM analysis.

HEK293 and HCT116 cells were able to proliferate in Glu +

-supplemented dFBS medium and were therefore pre-

cultivated for over one month for cell growth assays before

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Bayram et al. 10.3389/fmolb.2022.859787

being seeded at a density of 2E+6 cells on 10 cm plates. RKO cells

were unable to proliferate in Glu + NH

-supplemented medium

and were therefore pre-cultivated in Gln-supplemented dFBS

medium and changed to medium containing Glu + NH

performed 3 days prior to the SIRM experiment.

For SIRM experiments, cells were then labelled for 24 h with

C labelled glutamate and

N labelled ammonium and treated in

parallel with either 500 µM Methionine Sulfoximine (MSO,

Sigma Aldrich) or, as a negative control, sterile water (H

O).

For pSIRM experiments, cells were pre-treated for 6 h with either

1 mM MSO or, as a negative control, sterile water, in fresh Glu +

-supplemented dFBS medium. Afterwards, cells were

labelled for 15 min, 30 min, 1 h, and 3 h with

C labelled

glutamate and

N labelled ammonium, with or without 1 mM

MSO. In both experiments, SIRM and pSIRM, 1 mM

C labelled

glutamate was used. However, for SIRM experiments 96 µM

labelled ammonium were used, whereas for pSIRM 0.8 mM

labelled ammonium were used. Cells were harvested and

extracted in a methanol-chloroform-water solution as

described elsewhere [DOI: 10.1016/B978-0-12-801329-

8.00009-X].

Intracellular amino acids were measured as TBDMS

derivatives by high-resolution GC-MS. Dried cellular extracts

were mixed with 25 µl MTBSTFA (Sigma) and 25 µl ACN and

incubated at constant shaking for 1 h at 80°C. Derivatization was

automatized on a TriPlus RSH auto-sampler (Thermo Fisher)

and each sample was injected immediately after the

derivatization. Samples were injected into a Q Exactive GC

Orbitrap system (Thermo Fisher) with a splitof 1:5 (1 µl

injection volume) in a temperature-controlled injector

(TriPlus RSH auto-sampler, Thermo Fisher) with a bafﬂed

glass liner. The initial temperature was 80°C for 15 s, followed

by an increase of 7°C/s up to 260°C, which is held for 3 min at the

end of the temperature program. Gas chromatographic

separation was carried out on a Trace 1,300 GC (Thermo

Fisher) equipped with a TG-5SILMS column (30 m length,

250 µm inner diameter, 0.25 µm ﬁlm thickness (Thermo

Fisher). Helium was used as the carrier gas (1.2 ml/min ﬂow

rate). Gas chromatography was performed with an initial

temperature of 68°C for 2 min, followed by an increase of 5°C/

min up to 120°C, followed by an increase of 7°C/min up to 200°C,

followed by an increase of 12°C/min up to 320°C which is held for

6 min. The spectra were recorded in a mass range of m/z =

60––600 with resolution at 200 m/z set at 120,000.

The elemental composition of different fragments for

glutamine were calculated based on the exact mass and

compared with known literature-values. To extract the

intensities for the different isotopic masses we constructed a

compound library including the mass shifts induced by

C and

N. Mass shifts were calculated via a custom R-Script based on

the known masses for the fragments and the number of

potentially incorporated carbon and nitrogen atoms. Each

incorporated

Cor

N increased the target mass by

1.0033548 or 0.99693689, respectively.

For the SIRM experiment, samples were then processed and

peaks were integrated with Traceﬁnder 5.0 (Thermo Fisher), by

importing this target list as a Traceﬁnder Compound database

and extracting the extracted ion chromatograms (EIC) within a

5 ppm window. For the pSIRM experiment, samples were

processed and peaks integrated in Xcalibur Quanbrowser

(Thermo Fisher), extracting EIC with a mass tolerance of

2.5 ppm. For both, peak integration quality was visually

checked and ﬁnally all peak areas were exported.

2.6 Measurement of free nucleotides

Free nucleotides were measured by direct infusion MS on a Q

Exactive HF (Thermo Fisher) coupled to a Triversa Nanomate

(Advion) nanoESI ion source. The Triversa Nanomate was

operated in negative mode, with 1.5 kV spray voltage and

0.5 psi head gas pressure. The spectra were recorded for a

duration of 3 min in a mass range of m/z = 140–850 m/z

mass units with resolution at 200 m/z set at 240,000. A target

list with 48 compounds was prepared in a similar way as

described above. The M-H fragment was further calculated by

subtracting 1.00728 from the exact mass of the uncharged

molecule. For the extraction of the peak intensities the raw

ﬁles were ﬁrst converted to.dta2d ﬁles using TOPPAS

FileConverter tool (Kohlbacher et al., 2007a;Kohlbacher et al.,

2007b;Sturm et al., 2008).

The.dta2d ﬁles were then processed with a custom R script.

Brieﬂy, zero intensities and TIC intensities were removed from

the dataﬁles as well the ﬁrst and last ﬁve scans as these scans tend

to be instable. All masses that ﬁt into a 5 ppm window for each

mass in the target list was associated to that speciﬁc compound.

To extract only the apex the most intense mass per compound

and scan was kept. Finally, the median and the standard

deviation for all the scans was calculated to obtain a single

readout per compound and sample.

Natural abundance correction for both types of experiment

was performed using the Accucor package (URL: https://doi.org/

10.1021/acs.analchem.7b00396).

3 Results

3.1 Cell growth assay in fetal bovine serum

vs. dialyzed fetal bovine serum

Dejure and Royla, tested the effect of glutamine starvation on

GEO, HCT116, HEK293, HT29, RKO and SW480 cells and

found that all cell lines were not able to proliferate except

HEK293 (Dejure et al., 2017).

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Bayram et al. 10.3389/fmolb.2022.859787

We investigated as to whether two colon cancer cell lines

HCT116 and RKO can can adapt to glutamine depletion and

removed glutamine from the medium for several days (Figure 1).

Also in this experiment HEK293 cells exhibited glutamine

independence, but RKO and HCT116 cells were not able to

proliferate. In order to exclude that the glutamine independence

of HEK293 cells is not caused by small molecules provided by the

fetal bovine serum (FBS) we used dialyzed FBS and repeated the

proliferation experiment (Figure 2). Interestingly, in this case also

HEK293 cells were not able to proliferate when glutamine was

deprived. Thus, glutamine was also essential for HEK293 cells

when using dialyzed FBS.

3.2 Cell growth assay in supplemented

dialyzed fetal bovine serum

In order to identify which metabolic pathways are efﬁciently

utilised in glutamine-depleted condition, we monitored cell

survival and growth upon supplementation with substrates of

the glutamine-centric metabolic network. To achieve this, we

supplemented: Alanine (Ala), Ala + ammonium (NH

Asparagine (Asn), Aspartate (Asp), Asp + NH

, Glutamate

(Glu), Glu + NH

in dialyzed FBS medium (Figure 3).

Viable cell count was determined at every passage over the

course of 31 days. Cell count data were log2-transformed and

graphically represented. Cell doubling time was calculated based

on the division of culture duration by delta in log2-transformed

cell counts. All three tested cell lines exhibit the highest

proliferation rate and lowest doubling time in Gln-

supplemented medium (Figure 3). In HEK293 and

HCT116 cells the proliferation rate in Glu + NH

supplemented medium is close to that in Gln-supplemented

medium. For HCT116 cells proliferation in Asp + NH

supplemented media is remarkably high. In HEK293 cells also

the addition of glutamate leads to intermediate cell proliferation

rates. Contrary, RKO cells can not compensate glutamine

withdrawal under any condition. RKO cell viability decreased

and cell death occurred, preventing the possibility of obtaining

viable cell count data after 5 days onwards. Therefore, the

FIGURE 1

Cell growth assay for HEK293, HCT116, and RKO cells in non-dialyzed FBS with 2 mM or 0 mM glutamine (Gln) in the cell culture media. Cell

count was determined every 24 h over the course of 96 h and is shown relative to t= 0 as mean ± SD.

FIGURE 2

Cell growth assay for HEK293, HCT116, and RKO cells in dialyzed FBS with 2 mM or 0 mM glutamine (Gln) in the cell culture media. Cell count

was determined over the course of 7 days and is shown relative to t= 0 as mean ± SD.

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Bayram et al. 10.3389/fmolb.2022.859787

proliferation rate for Ala, Ala + NH

, Asn, Asp- Asp + NH

Glu, Glu + NH

-supplemented media is 0.

3.3 Methionine sulfoximine inhibitor

proliferation assay

The cell growth assays show that in glutamine-depleted

dialyzed FBS conditions, HEK293 and HCT116 cells

proliferate best when supplemented with the substrates of

GLUL: Glu and NH

(Figure 3). Based on this result, an

inhibitor assay was performed to assess the effect of blocking

de novo glutamine synthesis. Therefore, cells were treated with

MSO, a competitive inhibitor of GLUL. As RKO cells are unable

to proliferate in glutamine-depleted conditions, they were not

subjected to this assay.

A pilot experiment (data not shown) was performed in

HEK293 and HCT116 cells and an inhibitor concentration of

500 µM was found to be effective. The inhibitor-containing

medium was refreshed and viable cell count was determined

every 24 h over a 96 h time period. A parallel assay was

performed using water, the solvent control, instead of MSO.

Each measurement was taken from three biological replicates.

The mean and standard deviation of the viable cell counts for

each time point were graphically represented (Figure 4).

Untreated HEK293 and HCT116 cells exhibit similar

proliferation rates to those observed in the previous growth

assay for all tested different conditions. However, MSO-

treated HEK293 and HCT116 cells only show proliferation in

Gln-supplemented medium. Showing that the chosen inhibitor

concentration is not harmful to cells if glutamine is provided.

3.4 Targeted stable isotope resolved

metabolomics and methionine

sulfoximine-treatment

We designed a dual tracer stable isotope resolved

metabolomics (SIRM) study using

C and

N labelled

substrates (

C glutamate,

N ammonium) to determine

whether the cells utilise extracellular substrates for de novo

glutamine synthesis and subsequent nucleotide biosynthesis.

Based on the results of the cell growth assays, Glu + NH

supplemented medium was chosen to trace nitrogen and carbon

FIGURE 3

Cell Growth Assay in supplemented dialyzed FBS in HEK293, HCT116, and RKO cells. Cell growth upon the application of various amino acid

substrates: Alanine (Ala): 1 mM; Ala: 1 mM + NH

: 0.8 mM; Asparagine (Asn): 1 mM; Aspartate (Asp): 1 mM; Asp: 1 mM + NH

: 0.8 mM; Glutamate

(Glu): 1 mM; Glu: 1 mM + NH

: 0.8 mM; Glutamine (Gln): 2 mM. Viable cell count was determined at every passage over the course of 31 days. Cell

count data (each n= 2) were Log2-transformed and are shown as mean ± SD. The doubling time was calculated based on the duration in

culture and the number of duplications underwent during this time (i.e., duration/Δlog2(cell count)).

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Bayram et al. 10.3389/fmolb.2022.859787

incorporation into glutamine. The experiment was performed in

three biological replicates.

Glutamine can be synthesised by GLUL-mediated ligation of

glutamate and ammonium, with ammonium providing the

amido-group. The ligation of

-glutamate and

N-ammonium was monitored by GC-MS detection of

-, and

-glutamine isotopologues. Automated peak

extraction from GC-MS spectra was performed via Traceﬁnder

(Thermo Fisher) and mean

C and

N enrichment calculations

were performed using custom R scripts. In HCT116 and

HEK293 cells,

C- and

N-incorporation into glutamine was

detected in several characteristic glutamine-3TBDMS fragments,

as indicated by the corresponding

C- and

N-induced mass

shift of the peaks. Glutamine-3TBDMS fragments comprising a

5C skeleton and 2N atoms underwent a mass shift of

approximately 6 Da (m+6) while fragments comprising a 4C

skeleton and 2N atoms underwent a mass shift of 5 Da (m+5),

corresponding to

and

isotopologues,

respectively. The cleanest signal was obtained for the fragment

at 431 m/z and therefore this fragment was used for further

analysis.

In the pilot experiment HCT116 and HEK293 cells were

labelled for 24 h with 13C glutamate and 15N ammonium. In

HEK293 cells 51% enrichment of

C and 2% enrichment of

in the glutamine-3TBDMS fragment were monitored.

HCT116 cells have almost twice as much

C enrichment at

87% and

N enrichment at 22%. In both HEK293 and

HCT116 cells MSO treatment abolished

C and

enrichment to 0%. RKO cells do not demonstrate

Cor

enrichment in untreated and MSO-treated conditions (Figure 5).

The contribution of newly synthesised glutamine

isotopologues to nucleotide biosynthesis was monitored by

direct-infusion MS detection of nucleotide isotopologues. Peak

extraction from direct infusion-MS spectra was performed

manually with XCalibur Qualbrowser (Thermo Fisher). In the

de novo purine biosynthesis pathway, glutamine donates two

nitrogen atoms to IMP and AMP, and three nitrogen atoms to

GMP. HEK293 and HCT116 demonstrate

N enrichment in

AMP and GMP while RKO cells do not. In HEK293 and

HCT116 cells, one

N atom (N1) is incorporated into each

AMP and GMP. HEK293 cells exhibit 25% enrichment of

AMP and 20% enrichment of

-GMP, while HCT116 cells

FIGURE 4

Proliferation inhibition assay for HEK293, HCT116, and RKO cells. Investigation of cell growth upon the application of GLUL inhibitor MSO

(500 µM) or H

O with Alanine (Ala): 1 mM; Ala: 1 mM + NH

: 0.8 mM; Asparagine (Asn): 1 mM; Aspartate (Asp): 1 mM; Asp: 1 mM + NH

: 0.8mM;

Glutamate (Glu): 1mM; Glu: 1 mM + NH

: 0.8 mM; Glutamine (Gln): 2 mM. Cell count (each n= 2) was determined every 24 h over the course of

96 h and is shown relative to t= 0 as mean ± SD.

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Bayram et al. 10.3389/fmolb.2022.859787

exhibit 35% enrichment of

-AMP and 15% enrichment of

GMP (Figure 6).

In a second step we analyzed the dynamics of glutamine

synthesis in HCT116 and HEK293 cells in a time course

manner. The cell lines were incubated for 15 min, 30 min 1 h

and 3 h with

C labeled glutamate and

N labeled

ammonium. Label incorporation in glutamine was

analyzed as described above (Figure 7). Interestingly label

incorporation in glutamine can be found already after 15 min

in HCT116 cells and after 30 min in HEK293 cells. Both cell

lines show a fast glutamine synthesis but the kinetics are

different. In HCT116 cells the incorporation of

N labeled

ammonium into glutamine exceeds the formation of carbon

and nitrogen labeled glutamine, this argues for faster

ammonium import into HCT116 cells compared to

HEK293 cells.

Interestingly, we performed a western blot analysis to analyze

GLUL protein expression in the three cell lines and found that

GLUL protein is present in all cell line even under normal

conditions (Supplementary Material). We compared the

western blot result with proteomics data (not shown) and

could also ﬁnd speciﬁc peptides for GLUL in all cell lines.

Thus, the reason for the lacking GLUL activity in RKO cells

cannot be explained by missing GLUL protein levels but must be

caused by other reasons, e.g., mutations in the GLUL gene or

impaired transport of substrates for GLUL reaction.

4 Discussion

So far stable isotope tracing studies with multiple isotopic

tracers were performed without speciﬁc applications to

demonstrate how this technology can add more information,

compared to single isotope tracing methods. Here we show for

the ﬁrst time that this technology can be used to address speciﬁc

reactions in the metabolic network and to address clinically

relevant questions. In our study we analyzed the activity of

glutamine synthetase (GLUL) by applying both substrates

glutamate and ammonium labelled with stable isotopes.

Glutamine synthetase (GLUL), is of major interest, because

this enzyme may be a resistance factor in metabolic cancer

treatments; like the asparaginase treatment for acute

lymphoblastic leukemia (ALL) and solid cancer (Rotoli et al.,

2005). Glutamine is an important nutrient supporting cell growth

and proliferation, oncogenic mutations often render cancer cells

glutamine-dependent (Altman et al., 2016). In glutamine-

depleted conditions, α-ketoglutarate, aspartate and glutamate

supplementation have been demonstrated to rescue cell

growth of glutamine-dependent cancer cells to a certain

extend (Zhu et al., 2017).

In our study we analyzed the nature of glutamine

addiction of three selected cell lines. HEK293 cells were

partially glutamine auxotroph and HCT116 and RKO cells

glutamine addicted (Dejure et al., 2017). We found that the

FIGURE 5

C and

N enrichment in glutamine in untreated and MSO-treated HEK293, HCT116 and RKO Cells. Cells were cultivated with

C5-glutamate and

N-ammonium for 24 h. After obtaining mass spectra, peak areas were extracted and natural isotope abundance correction and isotope enrichment

calculations were performed. Data represent mean

C and

N enrichment (%).

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Bayram et al. 10.3389/fmolb.2022.859787

ability of HEK293 cells to proliferate under glutamine

deprived conditions did depend on the usage of non-

dialyzed FBS, if we used dialyzed serum also HEK293 cells

did depend on external glutamine supply. This demonstrates

that glutamine addiction is found in all tested cell lines in our

study.

In order to investigate the metabolic pathways that can

contribute to glutamine autotrophy we applied a selected set

of amino acids in a deﬁned knock out medium. Although these

conditions are artiﬁcial or synthetic compared to the natural

environment of a cancer cell, this experiment can be used to

understand the metabolic wires around glutamine (Figure 8); we

supplemented: Alanine (Ala), Ala + ammonium (NH4+),

Asparagine (Asn), Aspartate (Asp), Asp + NH4+, Glutamate

(Glu), Glu + NH4+ in dialyzed FBS medium. Because of the

absence of GLUL activity in RKO cells none of the supplements

could contribute to cell growth and survival. This result clearly

shows that all pathways that allow glutamine independency

funnel into de-novo glutamine synthesis via GLUL. Similarly,

the application of the speciﬁc GLUL inhibitor MSO abolished the

capacity of HEK293 and HCT116 cells to proliferate without

glutamine using the supplemented nutrients. Our results

demonstrate once more that GLUL is the major player in

glutamine independence.

FIGURE 7

Isotope incorporation into glutamine after pulse labelling with

-glutamate and

N-ammonium in HCT116 and HEK293 cells. HCT116 and

HEK293 cells were incubated with

-glutamate and

N-ammonium for 15 min, 30 min, 1 h, and 3 h (n= 2 each). Shown are the relative pool sizes

of non-labelled (C0N0),

N labelled (C0N1),

labelled (C5N0)

N-

labelled (C5N1) glutamine.

FIGURE 6

N Enrichment in AMP/GMP (purine nucleotides) in HEK293, HCT116 and RKO cells. Cells were cultivated with

-glutamate and

N-ammonium for

24 h. Nucleotide isotopologues were measured via direct-infusion MS and relative quantities are graphically represented. Data represent mean ± SD

of three biological replicates.

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Bayram et al. 10.3389/fmolb.2022.859787

Using high resolution mass spectrometry, we were able to

monitor GLUL activity via a dual-tracer targeted SIRM

approach. This method allowed us to measure a speciﬁed

reaction by the application of multiple isotopic tracers. We

observed in the pilot experiment a cell-line speciﬁc

incorporation of extracellular ammonium into glutamine:

HCT116 cells displayed a higher incorporation of

extracellular ammonium for glutamine synthesis than

HEK293 cells. However, we do not know if the available

ammonium is spent within 24 h. In subsequent experiments

the ammonium concentration was increased. In order to

retrieve dynamic information about the uptake rates of

individual substrates, a time course experiment was

performed. The time and stable isotope resolved

metabolomics experiments using multiple tracers delivered

valuable information about the metabolic activity facilitated

in the cell lines. The data show that HCT116 cells have a faster

uptake of glutamate and that internal ammonium is used

within the ﬁrst 15 min of the pulse experiment. The data

indicate that HCT116 cells possess higher

glutamine synthesis rates. Both cell lines show high levels

of stable isotope enrichment in glutamine after 30 min

labeling time.

By using direct-infusion MS, we detected

C and

enrichment in the de novo purine biosynthesis pathway in

HEK293 and HCT116 cells, when

C glutamate and

ammonium were supplied. To demonstrate the essentiality of

GLUL activity to de novo glutamine synthesis and downstream

nucleotide synthesis, inhibition of GLUL via MSO treatment

ablated

C and

N incorporation.

Interestingly, RKO cells do not demonstrate

C and

incorporation even in the absence of MSO treatment, indicating

that either GLUL is inactive or the import of these substrates is

compromised. The results from the dual-tracer targeted SIRM

study reﬂect the observations from the cell growth and inhibitor

assays (Figure 6). Taken together; the cell growth assays, inhibitor

studies and SIRM analyses reveal that, in glutamine depleted

conditions cell growth is dependent upon de novo glutamine

synthesis.

The usage of the dual isotope tracing strategy to measure

targeted and enzymatic activity in the cellular network in a

timeresolvedmannerisanadvantage.Thiswasnotdoneso

far, and our study is the ﬁrst showing the power of this

technology also for a clinically relevant question. We could

show at multiple layers that, despite glutamine synthetases is

expressed at the protein level, GLUL activity is absent in RKO

cells, thus we show that expression levels alone cannot

explain all metabolic activities.

The complex growth experiment highlights the role of an

active glutamine synthesis to rescue glutamine withdrawal by

using other amino acids, or a combination of amino acids and

ammonium. We could also show that in all the reactions that

we tested GLUL is the key enzyme and consequently; blocking

glutamine synthetase with the inhibitor MSO abolishess

growth and proliferation in the tested cell lines. Therefore,

we propose that this method can be applied in clinical studies

assessing different kinds of tumour cells and measuring

glutamine synthetase activity in vivo.

Overall, we show that concurrent stable isotope labelling

serves as a powerful tool for probing not only metabolic

FIGURE 8

Schematic of glutamine metabolism and MSO inhibition of glutamine synthetase (GLUL).

Frontiers in Molecular Biosciences frontiersin.org10

Bayram et al. 10.3389/fmolb.2022.859787

pathways, but also independent enzymatic reactions.

Leveraging this tool enabled us to validate our

observations from in vitro cell-based assays and

demonstrate an essential reaction underlying the capacity

of cells to adapt to glutamine-depletion. However, to detect

mass shifts induced by small molecules such as

Cand

atoms, a very high resolution is needed (Su et al., 2017). In the

absence of such a high resolution, mathematical models can

be used to calculate the relative contribution of these

molecules. We utilised the R package IsoCorrectoR to

calculate the relative contribution of

Cand

Nin

glutamine. For the purine nucleotides, we were able to

resolve

Cand

N incorporation from the raw data.

Our dual-tracer targeted SIRM study highlights the

potential for high resolution mass spectrometry to monitor

speciﬁc biological reactions at the atomic level. In future it

can be envisioned to study more enzymatic reactions using

concurrent isotope tracing techniques. We propose that all

metabolic reactions that require two or more substrates that

can be addressed with diverse isotopic labeling can be

analyzed using this method, e.g., reactions within the de

novo nucleotide biosynthesis or hexose amine biosynthesis.

This will be of mayor advantage if enzymatic activity essays

are not established.

Data availability statement

The raw data supporting the conclusion of this article

will be made available by the authors, without undue

reservation.

Author contributions

SB,YR,SG,andCVperformedexperiments.SB,YR,SG,

MF,MP,SF,GM,andSKanalyzedthedata.SB,YR,MF,MP,

and SK wrote manuscript. SK supervised the study.

Funding

SB was supported by the Sander Foundation and SF by DKTK

(German consortium for translational cancer research). MP was

funded by the Helmholtz association (AMPRO), YR and MF by

Deutsche Krebshilfe (Enable). SG was supported by the

Bundesministerium für Bildung und Forschung funding MSTARS

(Multimodal Clinical Mass Spectrometry to Target Treatment

Resistance).

Acknowledgments

We thank Jenny Grobe for excellent technical assistance.

Conﬂict of interest

The authors declare that the research was conducted in the

absence of any commercial or ﬁnancial relationships that could

be construed as a potential conﬂict of interest.

Publisher’s note

All claims expressed in this articlearesolelythoseoftheauthors

and do not necessarily represent those of their afﬁliated

organizations, or those of the publisher, the editors and the

reviewers. Any product that may be evaluatedinthisarticle,or

claim that may be made by its manufacturer, is not guaranteed or

endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can

be found online at: https://www.frontiersin.org/articles/10.3389/

fmolb.2022.859787/full#supplementary-material

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